Vector discovery and characterization An important aspect of the use of AAV vectors is the host immune response and the binding of ligands to the particle. Toward defining this structure-function relationship we have determined the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, to 3.45- resolution (Govindasamy 2013). The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between &#946;H and &#946;I of the core &#946;-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity. In 2003 we reported the development of a microarray based high throughput screening technique for identifying gene expression patterns that correlated with a specific phenotype (Di Pasquale et al. 2003). This approach was termed comparative gene analysis (CGA). Since our initial publication we have continued to refine the bioinformatics aspect of this approach and identified several receptors for different viruses including AAVs and filoviruses. This work has lead to a better understanding of the overall virus lifecyle of AAVs and lead to the reclassification of the family Parvoviridae (Cotmore et al. 2014). This work has also highlighted the role of protein glycosylation in the lifecycle of some viruses and suggested that alterations in host glycosylation could prevent virus attachment and maturation. In collaboration with the NIH undiagnosed diseases program, we were able to test this hypothesis in vivo by studying virus replication in cells isolated from patients with genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide) (Sadat et al. 2014). We evaluated virus replication and infection in two siblings with CDG-IIb. As expected both had a limited clinical history of any infectious diathesis. Our results show that these patients do not possess an altered susceptibility to Adenovirus type 5 or Poliovirus 1, non-enveloped viruses; or to Vaccinia, a glycosylation-independent for entry or egression enveloped virus. In contrast, they display a markedly reduced susceptibility to infection with HIV and influenza, two glycosylation-dependent enveloped viruses. While we cannot make broad generalizations, these data appear to suggest that altered glycosylation might modify infection susceptibility to viruses that require protein glycosylation to complete their infection cycle. AAV vectors have been tested in over 100 clinical trials with an excellent safety profile and clinical benefit in several diseases such hemophilia B, Lebers congenital amaurosis and lipoprotein lipase deficiency. However, poor transduction in some cell types has hindered the application of this technology to more conditions. To address this issue we have worked to develop a directed evolution approach to screening AAVs for optimal transduction activity. As a proof of concept we have applied this technology to developing vectors able to transduce primary human keratinocytes (Sallach et al 2013). All reported vectors lack efficient transduction activity on this cell type which represents an important target because of their ease of preparations and use in skin grafts. By making a library of AAV particles expressing distinct peptides, we then applied selective pressure to identify vectors with improved transduction activity. Selected AAVs engaged novel receptors that increased cell entry efficiency by up to 2,500- fold. The novel targeting vectors transduced human keratinocytes with high efficiency and a remarkable specificity even in mixed cultures of human keratinocytes and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. Application of this same technology should improve gene transfer to human salivary glands. Sjogrens syndrome Primary Sjgrens syndrome (pSS) is an autoimmune disease, characterized by lymphoid cell infiltration into the salivary and lacrimal glands, and affects 0.5% of the population in the United States of which 90% are women. The consequence of chronic immune cell activation in these exocrine glands is diminished secretory function, which leads to symptoms of dry mouth and dry eyes. In order to understand the environment of the salivary gland that might contribute to the gender bias associated with pSS we compared the transcriptome of male and female salivary glands from healthy individuals (Michael et al. 2012). Comparison of the transcriptome of minor salivary glands from normal male and female volunteers with that of salivary glands and other secretory epithelia identified a number of gender and tissue-specific gene expression patterns. These differences include, but are not limited to, a diverse set of genes involved in immune modulation, chemotactic control, inhibition of complement, metabolism, and neurogenesis. Analysis of these changes provides insight into the protective and predisposing molecular factors that may be involved in the development of pSS. Some of the gene changes observed in this study correlate with previously observed sexual dimorphisms in salivary gland function and also illustrate several new targets for further investigation. To identify epithelial changes in gene expression within female patients associated with the loss of gland function, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with pSS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. A significant increase in expression of BMP-6 was observed in RNA isolated from pSS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with pSS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in pSS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in pSS may not be the direct effect of autoantibodies or immune activation